Analytical methods and arrays for use in the same

ABSTRACT

The present invention relates to a method for identifying the skin sensitizer potency of a test agent, and arrays and analytical kits for use in such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.provisional patent application No. 62/410,206, filed Oct. 19, 2016,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to a combination therapy for thetreatment of solid malignant tumors, particularly to combinations oftalimogene laherparepvec, a BRAF inhibitor, and an immune checkpointinhibitor, wherein the combination therapy shows enhanced anti-tumoreffect.

BACKGROUND

Cancer remains one of the most deadly threats to human health.Worldwide, the number of melanoma cases is increasing at a rate that isfaster than that of any other human cancer (Roberts et al, Br. J.Dermatol (2002) 146:7-17). Melanoma accounts for less than 2 percent ofskin cancer cases but causes a majority of skin cancer deaths. Theclinical outcome of patients with melanoma is highly dependent on thestage at presentation. Early-stage melanoma can often be completelyremoved via surgery of the affected area. But once it has metastasized,it is much harder to treat. In most cases, it is not possible tocompletely eliminate or cure the cancer once it has metastasized.Depending upon where and how large the metastases are, treatment mayinvolve chemotherapy, surgery, gene therapy, immunotherapy, radiationtherapy, and combinations of these.

Talimogene laherparepvec is an oncolytic virus developed for thetreatment of advanced solid tumors. Talimogene laherparepvec wasapproved by the United States Food and Drug Administration (FDA) inOctober 2015 and by the European Commission in December 2015 for thetreatment of metastatic melanoma. Talimogene laherparepvec is derivedfrom a strain of herpes simplex virus type 1 (HSV-1) and has beengenetically engineered to attenuate neurotropic and infectiousproperties while increasing selectivity for malignant rather thanhealthy cells. The virus has also been modified to producegranulocyte-macrophage colony-stimulating factor (GM-CSF), aninflammatory cytokine which is a chemo-attractant for various immunecells.

Despite recent success in the development of new therapies for melanoma,the vast majority of patients eventually succumb to their disease;hence, there remains a significant unmet need for improvement ofoutcomes in solid malignant tumor patient populations, including morespecifically the melanoma patient population.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method fortreating a tumor (e.g., a solid malignant tumor) or cancer in a subjectcomprising: administering to the subject a therapeutically effectiveamount of a BRAF inhibitor, an oncolytic virus, and an immune checkpointinhibitor.

In some embodiments, the oncolytic virus is an HSV-1-based oncolyticvirus. In specific embodiments, the oncolytic virus is talimogenelaherparepvec.

In further embodiments, the immune checkpoint inhibitor is selected froma small molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, and combinationsthereof. In certain embodiments, the immune checkpoint inhibitor is aPD-1 antagonist. In specific embodiments, the immune checkpointinhibitor is an anti-PD-1 antibody.

In certain embodiments, the administration of the immune checkpointinhibitor is continued until it induces tumor regression, remission, oreradication.

In some embodiments, the administration of each BRAF inhibitor,oncolytic virus, and the immune checkpoint inhibitor is continued untilit induces tumor regression, remission, or eradication.

In certain embodiments, the BRAF inhibitor is selected from the groupconsisting of PLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436(dabrafenib), GDC-0879, and PLX4032 (vemurafenib), or a derivativethereof. In a specific embodiment, the BRAF inhibitor is vemurafenib.

In some embodiments, the tumor (e.g., solid malignant tumor) or canceris a BRAF mutant tumor or cancer. In some embodiments, the BRAF mutanttumor or cancer is melanoma, thyroid cancer, brain cancer, lung cancer,gastric cancer, or colon cancer. In certain embodiments, the BRAF mutanttumor (e.g., solid malignant tumor) or cancer is melanoma.

In certain embodiments, the present invention relates to method fortreating melanoma (e.g., BRAF mutant melanoma) in a subject comprising:administering to the subject a therapeutically effective amount of BRAFinhibitor, an oncolytic virus, and an immune checkpoint inhibitor.

In some embodiments, the oncolytic virus is an HSV-1-based oncolyticvirus. In specific embodiments, the oncolytic virus is talimogenelaherparepvec.

In further embodiments, the immune checkpoint inhibitor is selected froma small molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, and combinationsthereof. In some embodiments, the immune checkpoint inhibitor is a PD-1antagonist. In specific embodiments, the immune checkpoint inhibitor isan anti-PD-1 antibody.

In some aspects, administration of the checkpoint inhibitor is continueduntil it induces melanoma tumor regression, remission, or eradication.In further aspects, the administration of each BRAF inhibitor, oncolyticvirus, and checkpoint inhibitor is continued until it induces melanomatumor regression, remission, or eradication.

In some embodiments, the BRAF inhibitor is selected from the groupconsisting of PLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436(dabrafenib), GDC-0879, and PLX4032 (vemurafenib), or a derivativethereof.

In certain embodiments, the present disclosure relates to a method ofreducing CD4+FOXP3+ cellular population within a tumor (e.g., solidmalignant tumor), the method comprising delivering to the tumor aneffective amount of BRAF inhibitor, an oncolytic virus, and a checkpointinhibitor.

In certain embodiments, the immune checkpoint inhibitor is selected froma small molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2. PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049 and combinationsthereof. In some embodiments, the immune checkpoint inhibitor is a PD-1antagonist. In specific embodiments, the immune checkpoint inhibitor isan anti-PD-1 antibody.

In some embodiments, the BRAF inhibitor is selected from the groupconsisting of PLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436(dabrafenib), GDC-0879, and PLX4032 (vemurafenib), or a derivativethereof.

In certain embodiments, the present invention provides a method ofstimulating an immune response in a subject (e.g., a subject with a BRAFmutant tumor such as, e.g., melanoma) comprising: administering to thesubject a therapeutically effective amount of a BRAF inhibitor, anoncolytic virus, and a checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected from asmall molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2. PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, and combinationsthereof. In further embodiments, the immune checkpoint inhibitor is aPD-1 antagonist. In specific embodiments, the immune checkpointinhibitor is an anti-PD-1 antibody.

In some embodiments, administration of each BRAF inhibitor, oncolyticvirus, and checkpoint inhibitor is continued until stimulation of theimmune response is achieved.

In certain embodiments, BRAF inhibitor is selected from the groupconsisting of PLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436(dabrafenib), GDC-0879, and PLX4032 (vemurafenib), or a derivativethereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a timeline and scheme for the administration of treatments tomice of the desired genotype (i.e., mice which are heterozygous BRAF(BRAFF-V600E/+), homozygous for floxed PTEN (PTENF−/−) andTyr::CreERT2). As illustrated, mice with the desired genotype wereraised until they were 6-8 weeks old, then underwent tumor induction,were treated 4-6 weeks post tumor induction, and were sacrificed at theend of the study.

FIG. 2 is a graph of tumor volume (y-axis) vs days of treatment measuredin six distinct treatment groups: (1) group 1 received control chow,isotype control 2A3, and PBS (phosphate buffered saline) (line 1); (2)group 2 received BRAF inhibitor PLX 4720 containing chow (BRAFi chow),isotype control 2A3, and PBS (line 2); (3) group 3 received BRAFi chow,anti-PD-1 antibody, and PBS (line 3); (4) group 4 received BRAFi chow,isotype control 2A3, and OncoVex^(mGM-CSF) (line 4); (5) group 5received BRAFi chow, anti-PD-1 antibody, and OncoVex^(mGM-CSF) (line 5);and (6) group 6 received control chow, anti-PD-1 antibody, andOncoVex^(mGM-CSF) (6).

FIG. 3 is a Kaplan-Meier survival curve of tumor-bearing animals treatedwith the following: (1) control chow, isotype control 2A3, and PBS (alsoreferred to herein as Control Group; line 1); (2) BRAF inhibitor PLX4720 containing chow (BRAFi chow), isotype control 2A3, and of PBS (alsoreferred to herein as BRAFi only; line 2); (3) BRAFi chow, anti-PD-1antibody, and PBS (also referred to herein as BRAFi+α-PD1; line 3); (4)BRAFi chow, isotype control 2A3, and OncoVex^(mGM-CSF) (also referred toas herein as BRAFi+OncoVex^(mGM-CSF); line 4); (5) BRAFi chow, anti-PD-1antibody, and OncoVex^(mGM-CSF) (also referred to herein asBRAFi+α-PD1+OncoVe^(mGM-CSF); line 5); and (6) control chow, anti-PD-1antibody, and OncoVex^(mGM-CSF) (also referred to herein asα-PD1+OncoVex^(mGM-CSF); line 6).

FIG. 4 expands upon the data in FIGS. 2 and 3 and represents tumorgrowth curves, survival curves, and ulceration rates between treatmentgroups. FIG. 4A is the mean tumor volume comparison of all mice upthrough 33 days. FIG. 4B is the mean tumor volume comparison of mice ingroups receiving BRAFi up through 57 days. A 2-way ANOVA analysis wasperformed comparing group 2 (BRAFi only) to other BRAFi treatment groupsand found significant tumor growth inhibition in group 5(BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p=0.0141). FIG. 4C is the survivalcomparison of mice in each treatment group using the log-rank(Mantel-Cox) test. Findings show prolonged survival for group 2 (BRAFionly) (p<0.0001), group 3 (BRAFi+α-PD1) (p=0.0002), group 4(BRAFi+OncoVex^(mGM-CSF)) (p<0.0001), and group 5(BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p<0.0001) in comparison to group 1(control) mice. FIG. 4D shows comparison of survival in treatment groupsreceiving BRAFi using the log-rank (Mantel-Cox) test. A trend towardsignificance in group 4 (BRAFi+OncoVex^(mGM-CSF)) (p=0.1218) and group 5(BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p=0.1561) when comparing to group 2(BRAFi only) mice was observed. FIG. 4E shows that tumor ulceration leadto stoppage of intratumoral injections. The number of mice receivingeach treatment was evaluated using two-tailed fisher's exact test. Thenumber of mice ulcerated was higher for those receivingOncoVex^(mGM-CSF) vs. not receiving OncoVex^(mGM-CSF) SF (p=0.0003),while comparison of BRAFi and anti-PD1 treatment was not significant.FIG. 4F also shows the size of tumors at timepoints when intratumoralinjections were stopped due to ulceration in each group was evaluated.The size of tumor was significantly smaller in group 5(BRAFi+α-PD1+OncoVex_(mGM-CSF)) (p=0.0214) and group 6(α-PD1+OncoVex^(mGM-CSF)) (p=0.0074) when compared to group 1 (ControlGroup) mice (ns>0.05, *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001).

FIG. 5 is a scatter plot showing significantly decreased percent ofCD4+Foxp3+ cells following the treatment of tumor-bearing animals with(1) control chow, isotype control 2A3, and PBS (Control); (2) BRAFinhibitor PLX 4720 containing chow (BRAFi chow), isotype control 2A3,and PBS (BRAFi Only); (3) BRAFi chow, anti-PD-1 antibody, and PBS(BRAFi+anti-PD1); (4) BRAFi chow, isotype control 2A3, andOncoVex^(mGM-CSF) (BRAFi+OncoVex^(mGM-CSF)); (5) BRAFi chow, anti-PD-1antibody, and OncoVex^(mGM-CSF) (BRAFi+anti-PD1+OncoVex^(mGM-CSF)); and(6) control chow, anti-PD-1 antibody, and OncoVex^(mGM-CSF)(anti-PD1+OncoVex^(mGM-CSF)).

FIG. 6 expands upon the data in FIG. 5 and is the flow cytometryevaluation of intratumoral T Cell Infiltration. FIG. 6A is the percentof CD8+/CD3+ cytotoxic T lymphocytes (CTLs) in all 6 treatment groups.CTLs were found to be increased in group 3 (BRAFi+α-PD1) (p=0.0002)(p=0.0476), group 4 (BRAFi+OncoVex^(mGM-CSF)) (p=0.0004), group 5(BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p=0.0001), and group 6(α-PD1+OncoVex^(mGM-CSF)) (p=0.0001) when compared to group 1 (control).FIG. 6B shows that CTL infiltration was highest in groups receivingOncoVex^(mGM-CSF) when compared to groups not receivingOncoVex^(mGM-CSF) (p<0.0001). FIG. 6C shows the percent ofCD4+FOXP3+/CD4+ regulatory T-cells (Tregs) in all 6 treatment groups.Infiltration of Tregs is decreased in group 4 (BRAFi+OncoVex^(mGM-CSF))(p=0.0082), group 5 (BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p<0.0001), andgroup 6 (α-PD1+OncoVex^(mGM-CSF)) (p=0.0031) when compared to group 1(control). FIG. 6D shows that Treg infiltration is lowest in groupsreceiving OncoVex^(mGM-CSF) when compared to groups not receivingOncoVex^(mGM-CSF) (p<0.0001). (ns>0.05, *≤0.05, **≤0.01, ***≤0.001,****<0.0001).

DETAILED DESCRIPTION

In one aspect, the present disclosure provides a method of treating atumor (e.g., a solid malignant tumor) or cancer, the method comprisingadministering to a patient, a therapeutically effective amount of BRAFinhibitor (“BRAFi”), an oncolytic virus, and an immune checkpointinhibitor. In one embodiment, the oncolytic virus is derived from aherpes simplex virus. In another aspect, the present disclosure providesa method of treating a tumor (e.g., a solid malignant tumor) or cancer,the method comprising administering to a patient, a therapeuticallyeffective amount of BRAF inhibitor, an HSV-1 based oncolytic virus(e.g., talimogene laherparepvec), and an immune checkpoint inhibitor.

The present disclosure also provides a method of treating melanoma(e.g., BRAF mutant melanoma), the method comprising administering to apatient, a therapeutically effective amount of BRAF inhibitor, anoncolytic virus, and an immune checkpoint inhibitor. In one embodiment,the oncolytic virus is derived from a herpes simplex virus.Additionally, the present disclosure provides a method of treatingmelanoma (e.g., BRAF mutant melanoma), the method comprisingadministering to a patient, a therapeutically effective amount of BRAFinhibitor, an HSV-1 based oncolytic virus (e.g., talimogenelaherparepvec), and an immune checkpoint inhibitor.

The present disclosure provides a combination therapy for treatingvarious types of tumors (e.g., solid malignant tumors) including BRAFmutant tumors. Particularly, the present disclosure providescompositions and methods combining an effective amount of BRAFinhibitor, an oncolytic virus, and an immune checkpoint inhibitor. Inone embodiment, the oncolytic virus is derived from a herpes simplexvirus. The present disclosure also provides compositions and methodscombining an effective amount of BRAF inhibitor, an HSV-1 basedoncolytic virus (e.g., talimogene laherparepvec), and a checkpointinhibitor. In some embodiments, the combination therapy demonstratessignificant enhancement of the anti-tumor effect compared to eitheragent alone. In particular embodiments, the enhancement of theanti-tumor effect is synergistic.

In addition, the present disclosure provides pharmaceutical compositionscomprising an effective amount of BRAF inhibitor, an oncolytic virus,and a checkpoint inhibitor. In one embodiment, the oncolytic virus isderived from a herpes simplex virus. The present disclosure alsoprovides pharmaceutical compositions comprising an effective amount ofBRAF inhibitor, an HSV-1 based oncolytic virus (e.g., talimogenelaherparepvec), and a checkpoint inhibitor.

The present disclosure is based in part on the unexpected discovery thatcombination of BRAF inhibitor, a herpes simplex virus based oncolyticvirus (e.g., talimogene laherparepvec), and an immune checkpointinhibitor (e.g., a PD-1 inhibitor) shows a synergistic inhibiting effecton formation and progression of cancer. This phenomenon was observed ina spontaneous in vivo model of melanoma (Examples 2 and 3). Thus, it wasshown that the combination of the present disclosure possessesbeneficial therapeutic properties, e.g. synergistic interaction, and astrong in-vivo anti-tumor response, which can be used as a treatment.These characteristics render it particularly useful for the treatment ofcancers and tumors (e.g., solid malignant tumors) including BRAF mutantcancers and tumors.

Thus, in particular embodiments, the present disclosure provides amethod of treating tumors (e.g., solid malignant tumors) or cancers,including BRAF mutant tumors or cancers, the method comprisingadministering to a patient, a therapeutically effective amount of BRAFinhibitor, an oncolytic virus, and a PD-1 inhibitor. For example, thepresent disclosure provides a method of treating tumors (e.g., solidmalignant tumors), including BRAF mutant tumors, the method comprisingadministering to a patient, a therapeutically effective amount of BRAFinhibitor, a herpes simplex virus based oncolytic virus (e.g.,talimogene laherparepvec), and a PD-1 inhibitor.

In one embodiment, the PD-1 inhibitor is an anti PD-1 antibody. In aspecific embodiment, the anti-PD-1 antibody is pembrolizumab (MK-3475).In another embodiment, the anti-PD-1 antibody is nivolumab (MDX 1106/BMS936558).

In one embodiment, the BRAF inhibitor is PLX4720. While not being boundby theory, it is expected that the benefit of the methods describedherein will be most pronounced for BRAF mutant tumors. Thus, in certainembodiments, the tumors will be screened prior to treatment for BRAFmutations and tumors positive for BRAF mutations will be treated asdescribed herein.

Accordingly, the present disclosure provides a method of treating tumors(e.g., solid malignant tumors), including BRAF mutant tumors, the methodcomprising administering to a patient, a therapeutically effectiveamount of BRAF inhibitor PLX4720, talimogene laherparepvec, and ananti-PD-1 antibody. In a particular embodiment, the solid malignanttumor is melanoma (e.g., BRAF mutant melanoma).

Definitions

As used herein, the term “immune checkpoint inhibitor” refers tomolecules that completely or partially reduce, inhibit, interfere withor modulate one or more checkpoint proteins. Checkpoint proteinsregulate T-cell activation or function. Numerous checkpoint proteins areknown, such as, Programmed Death 1 (PD-1) with its ligands PD-L1 andPD-L2, Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) and its ligands CD80and CD86, B7-H1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,2B4 (belongs to the CD2 family of molecules and is expressed on all NK,γδ, and memory CD8⁺ (αβ) T cells), OX-40, CD137. CD40, CD 160 (alsoreferred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases. A2aR andvarious B-7 family ligands (Pardoll, Nature Reviews Cancer 12: 252-264,2012). These proteins are responsible for co-stimulatory or inhibitoryinteractions of T-cell responses. Immune checkpoint proteins regulateand maintain self-tolerance and the duration and amplitude ofphysiological immune responses. Examples of immune checkpoint inhibitorsinclude antibodies or molecules/agents derived from antibodies.

As used herein, the terms “patient” or “subject” are usedinterchangeably and mean a mammal, including, but not limited to, ahuman or non-human mammal, such as a bovine, equine, canine, ovine, orfeline. Preferably, the patient is a human.

The term “melanoma” is used in the broadest sense and refers to allstages and all forms of cancer arising from melanocytes. The methods ofthe present invention can be used to treat several different stages ofcancer (e.g., melanoma). Most staging systems include informationrelating to whether the cancer has spread to nearby lymph nodes, wherethe tumor is located in the body, the cell type, whether the cancer hasspread to a different part of the body, the size of the tumor, and thegrade of tumor (i.e., the level of cell abnormality the likelihood ofthe tumor to grow and spread). For example, Stage 0 refers to thepresence of abnormal cells that have not spread to nearby tissue—i.e.,cells that may become a cancer. Stage I, Stage II, and Stage III cancerrefer to the presence of cancer. The higher the Stage, the larger thecancer tumor and the more it has spread into nearby tissues. Stage IVcancer is cancer that has spread to distant parts of the body. In aparticular example, the stage of melanoma is Stage IIIc to IV melanoma.

Melanoma is typically a malignant tumor associated with skin cancer.

As used herein, the terms “reduce or inhibit” is meant the ability tocause an overall decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to thesymptoms of the disorder being treated, the presence or size ofmetastases, or the size of the primary tumor.

As used herein, the phrases “treating cancer” and “treatment of cancer”and “treatment of tumors” mean to decrease, reduce, or inhibit thereplication of cancer cells; decrease, reduce or inhibit the spread(formation of metastases) of cancer; decrease tumor size; decrease thenumber of tumors (i.e. reduce tumor burden); lessen or reduce the numberof cancerous cells in the body; prevent recurrence of cancer aftersurgical removal or other anti-cancer therapies; or ameliorate oralleviate the symptoms of the disease caused by the cancer.

As used herein, the term “synergistic” refers to an interaction of aBRAF inhibitor and an oncolytic virus (e.g., talimogene laherparepvec)with an immune checkpoint inhibitor wherein the observed effect (e.g.,reduction of tumor volume) in the presence of the combination ofcompounds together is higher than the sum of the individual effects ofeach compound administered separately. In one embodiment, the observedcombined effect of the compounds is significantly higher than the sum ofthe individual effects.

As used herein, the term “BRAF inhibitor” refers to a compound or agentthat inhibits, decreases, lowers, or reduces at least one activity ofany of the isoforms or mutants of BRAF kinase. Examples of BRAFinhibitors include, but are not limited to, GSK2118436 (dabrafenib),PLX4720, and PLX4032.

As used herein, the term “anti PD-1 antibody” refers to an antibody thatinhibits PD-1 activity by, e.g., inhibiting the interaction between PD-1and its ligands (e.g., PD-L1 and PD-L2) and, thus, can enhance T-cellresponses and can mediate antitumor activity. The term “inhibits theactivity” relates to a decrease (or reduction) in lymphocyteproliferation or activity that is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more. PD-1-mediated activity can bedetermined quantitatively using T cell proliferation assays known in theart.

As used herein, the term “effective amount” refers to the quantity of acomponent that is sufficient to treat a subject without undue adverseside effects (such as toxicity, irritation, or allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of this invention, i.e. a therapeutically effective amount. Thespecific effective amount will vary with such factors as the particularcondition being treated, the physical condition of the patient, the typeof mammal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compounds or its derivatives.

The term “CD4+Foxp3+T regulatory cells (Tregs)” used herein refers toregulatory T cells expressing a Foxp3 protein. Foxp3 is expressed byCD4+CD25+ Tregs, and gain-of-function, overexpression and analysis ofFoxp3-deficient Scurfy (sf) mice show Foxp3 is essential to thedevelopment and maintenance of murine Tregs.

The combination therapy described in the present disclosure can be usedfor the treatment of various types of solid malignant tumors (e.g., BRAFmutant tumors). Non-limiting examples of preferred types ofcancers/tumors (including BRAF mutant tumors) for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer,lung cancer (e.g. non-small cell lung cancer), esophageal cancer,squamous cell carcinoma of the head and neck, liver cancer, ovariancancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia,lymphoma, and other neoplastic malignancies, and more generally solidtumors. Additionally, the present disclosure includes refractory orrecurrent malignancies whose growth may be inhibited using thecombinations described herein. In specific embodiments, the combinationtherapy described in the present disclosure can be used for thetreatment of BRAF mutant tumors. Such BRAF mutant tumors include, e.g.,melanoma, thyroid cancer, brain cancer, lung cancer, gastric cancer, andcolon cancer. In a particular embodiment, the BRAF mutant tumor ismelanoma. In other embodiments, the combination therapy described in thepresent disclosure can be used for the treatment of phosphatase andtensin homolog (PTEN) negative tumors.

Uses of the Combination Therapy

The combination therapy described in the present disclosure isparticularly advantageous because the anti-tumor effect of thecombination is enhanced compared to the effect of each compound alone,or the effects of a combination of any two compounds selected from BRAFinhibitor, oncolytic virus (e.g., talimogene laherparepvec), and a PD-1inhibitor. Moreover, it is anticipated that the dosage of each agent ina combination therapy can be reduced as compared to monotherapy witheach agent, while still achieving an overall anti-tumor effect. Inaddition, due to the synergistic effect, the total amount of compoundsadministered to a patient can advantageously be reduced, which mayresult in decreased side effects.

Enthusiasm relating to the potential of combinations of BRAF inhibitorswith checkpoint inhibitors (e.g., ipilimumab) has been historicallymuted in light of the prevailing viewpoint that the two agents were tootoxic for administration in combination to patients. The presentinvention demonstrates however that BRAF inhibitors can be safelyadministered in combination with some checkpoint inhibitors, such asanti-PD-1 compounds (e.g., anti-PD-1 antibodies).

The antibody or antigen binding fragments of the anti-PD-1 or immunemodulating antibody, in combination with an oncolytic virus (e.g.,talimogene laherparepvec) and a BRAF inhibitor can be used to increase,enhance, stimulate or up-regulate an immune response. The combinationtreatments described herein are particularly suitable for treatingsubjects having a disorder that can be treated by increasing the T-cellmediated immune response. Preferred subjects include human patients inneed of enhancement of an immune response.

T regulatory cells (Tregs) play an important role in the induction andmaintenance of immunologic tolerance (Malek and Castro, Immunity. 2010Aug. 27; 33(2): 153-165). In many human cancers and mouse models oftumor growth, the frequency of Treg and their suppressor functions areincreased as compared to those reported for healthy subjects. Moreover,there is considerable experimental and clinical evidence suggestingTregs are engaged in suppression of antitumor immune responses and thuscontribute to tumor escape from the host immune system (Droese et al.BMC Cancer. 2012; 12:134).

Two main types of regulatory T cells have been identified—natural andinduced (or adaptive)- and both play significant roles in tuning downeffector immune responses. The expression of surface markers such asCD25 on the cell surface and intracellular forkhead transcription factor(FOXP3) has been used to differentiate between these two Treg subsets byflow cytometry. Adaptive CD4(+)Foxp3(+) regulatory T (iTregs) expressingthe Foxp3 have been shown to suppress T cell-mediated host immuneresponses against self- and nonself-antigens. In several different typesof cancer, increased numbers of CD4+FOXp3+ Tregs have been associatedwith poor prognosis. For example, high CD4+Foxp3+ Tregs infiltration issignificantly associated with shorter overall survival (OS) in themajority of solid tumors including cervical, renal, melanoma, and breastcancer (Shang et al. Sci Rep. 2015 Oct. 14; 5:15179).

The combination therapy described in the present disclosure isadditionally suitable for reducing the CD4⁺FOXP3⁺ cellular populationwithin tumors (e.g., solid malignant tumors), including BRAF mutanttumors, such as in a method comprising administering a therapeuticallyeffective amount of BRAF inhibitor, an oncolytic virus (e.g., talimogenelaherparepvec), and an immune checkpoint inhibitor. Accordingly, thecombination therapy of the present disclosure can be used for thetreatment of a Foxp3+T regulatory cell (Treg) related disease. TheFoxp3+ Treg related disease may be a cancer or a tumor. As disclosedherein, the percentage of CD4+Foxp3+ Tregs was significantly lower incells receiving anti-PD-1 therapy. OncoVex^(mGM-CSF) and BRAFinhibition, compared with those that received only OncoVex^(mGM-CSF) andBRAF inhibition combined therapy or anti-PD-1 and OncoVex^(mGM-CSF) GMFcombination (Example 3). OncoVex^(mGM-CSF) is an HSV-1 modified in thesame manner as talimogene laherparepvec except that it contains the geneencoding murine GM-CSF instead of human GM-CSF.

The administration of the immune checkpoint inhibitor is continued untilit induces tumor regression or eradication. Furthermore, the duration ofeach component of the combined treatment disclosed here will continueuntil it induces tumor regression or eradication.

Furthermore, the present disclosure relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of (i) anoncolytic virus (e.g., an HSV-1 such as talimogene laherparepvec), (ii)a BRAF inhibitor or a pharmaceutically acceptable salt thereof, and(iii) a PD-1 inhibitor, or a pharmaceutically acceptable salt thereof.Alternatively, the present disclosure relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of (i) anoncolytic virus (e.g., an HSV-1 such as talimogene laherparepvec), and(ii) a BRAF inhibitor or a pharmaceutically acceptable salt thereof. Inanother aspect, the present disclosure encompasses a pharmaceuticalcomposition comprising a therapeutically effective amount of (i) anoncolytic virus (e.g., an HSV-1 such as talimogene laherparepvec), and(ii) a PD-1 inhibitor, or a pharmaceutically acceptable salt thereof. Infurther aspect, the present disclosure encompasses a pharmaceuticalcomposition comprising a therapeutically effective amount of (i) a BRAFinhibitor or a pharmaceutically acceptable salt thereof, and (ii) a PD-1inhibitor, or a pharmaceutically acceptable salt thereof. Alternatively,a pharmaceutical composition of the present disclosure may comprise (i)an oncolytic virus (e.g., an HSV-1 such as talimogene laherparepvec), or(ii) a BRAF inhibitor or a pharmaceutically acceptable salt thereof, or(iii) a PD-1 inhibitor, or a pharmaceutically acceptable salt thereof.In certain embodiments, a PD-1 inhibitor is an anti-PD-1 antibody. Infurther embodiments, anti-PD-1 antibody is nivolumab or pembrolizumab.In one embodiment, a BRAF inhibitor is PLX4720.

The various components of the combination therapy described in thepresent disclosure may be administered concurrently with, or separatelyfrom, other components in a treatment regimen. Additionally, thecombination therapy described herein further comprises administering achemotherapeutic agent, targeted therapy or radiation to the subjecteither prior to, simultaneously with, or after treatment with thecombination therapy. In an additional aspect, the tumor may be resectedprior to the administration of the BRAF inhibitor, an oncolytic virus(e.g., talimogene laherparepvec), and a PD-1 inhibitor.

The components of the combination therapy described herein can beadministered simultaneously, separately or sequentially.

Oncolytic Viruses

Advances in cancer vaccines and immunotherapy have generated aconsiderable interest in viral therapy such that “oncolytic viruses” maybe exploited to potentiate an anti-tumor immune response by exposing theimmune system to tumor antigens.

Pre-clinical and clinical studies evaluating the potential of severaloncolytic viruses are ongoing. Thus, in one aspect, the oncolytic virusof the current invention is CAVATAK™ (a coxsackievirus A21 (CVA21)),HF-10 (spontaneously mutated HSV-1), ONCOS-102 (engineered humanserotype 5 adenovirus), enadenotucirev (A11/Ad3 chimeric Group Badenovirus), genetically engineered strains of vesicular stomatitisvirus, REOLYSIN® (human reovirus), pexastimogene devacirepvec (vacciniapoxvirus), NDV-GM-CSF (newcastle disease virus encoding GM-CSF), BV-2711(HSV-1-based virus). PSI-001, VCN-01 (adenovirus expressing PH20hyaluronidase), OrienX-010 (HSV-1 expressing GM-CSF), or Ad-RTS hIL-12(adenovirus vector engineered to express human IL-12).

In some embodiments, the oncolytic virus is a herpes simplex virus. Inother embodiments, the oncolytic virus is a herpes simplex virus type 1(HSV-1). In yet other embodiments, the HSV-1 lacks functional ICP34.5genes, lacks a functional ICP47 gene and comprises a gene encoding humanGM-CSF. The ICP34.5 gene is located in the terminal repeats of the longregion of HSV and therefore there are two copies per genome. Previousstudies have shown that functional deletion of the neurovirulence gene,ICP34.5, renders the virus avirulent. The ICP47 gene is located in theunique short region of HSV. The ICP47 gene product interacts with thetransporter associated with antigen processing (TAP1 and TAP2) andblocks antigen processing via MHC class I molecules. Deletion of theICP47 gene allows greater antigen processing within infected cells andis intended to result in a concomitant increase in the immune responseto infected cells. GM-CSF is a cytokine known to be involved in thestimulation of immune responses. Using homologous recombination withplasmid DNA, heterologous genes, such as that encoding human GM-CSF, canbe inserted into the HSV viral genome, and viral genes, such as ICP34.5and ICP47, can be functionally deleted. In one embodiment, the herpessimplex virus is talimogene laherparepvec.

Talimogene laherparepvec is derived from HSV-1, an enveloped,double-stranded DNA virus which replicates in the nuclei of infectedcells. HSV-1 induces cell lysis resulting in the spread of viral progenyto nearby cells (Roizman et al. Lippincott Williams &Wilkins:Philadelphia. p. 1823-1897 (2013). Talimogene laherparepvec, HSV-1[strain JS1] ICP34.5-/ICP47-/hGM-CSF, (previously known asOncoVex^(mGM-CSF)), is an intratumorally delivered oncolyticimmunotherapy comprising an immune-enhanced HSV-1 that selectivelyreplicates in solid tumors. (Lui et al., Gene Therapy, 10:292-303, 2003;U.S. Pat. Nos. 7,223,593 and 7,537,924). The HSV-1 was derived fromStrain JS1 as deposited at the European collection of cell cultures(ECAAC) under accession number 01010209. In talimogene laherparepvec,the HSV-1 viral genes encoding ICP34.5 have been functionally deleted.Functional deletion of ICP34.5, which acts as a virulence factor duringHSV infection, limits replication in non-dividing cells and renders thevirus non-pathogenic. The safety of ICP34.5-functionally deleted HSV hasbeen shown in multiple clinical studies (MacKie et al, Lancet 357:525-526, 2001; Markert et al, Gene Ther 7: 867-874, 2000; Rampling etal, Gene Ther 7:859-866, 2000; Hunter et al, J Virol Aug; 73(8):6319-6326, 1999). In addition. ICP47 (which blocks viral antigenpresentation to major histocompatibility complex class I and IImolecules) has been functionally deleted from talimogene laherparepvec.Functional deletion of ICP47 also leads to earlier expression of US11, agene that promotes virus growth in tumor cells without decreasing tumorselectivity. The coding sequence for human GM-CSF, a cytokine involvedin the stimulation of immune responses, has been inserted into the viralgenome of talimogene laherparepvec. The insertion of the gene encodinghuman GM-CSF is such that it replaces nearly all of the ICP34.5 gene,ensuring that any potential recombination event between talimogenelaherparepvec and wild-type virus could only result in a disabled,non-pathogenic virus and could not result in the generation of wild-typevirus carrying the gene for human GM-CSF. The HSV thymidine kinase (TK)gene remains intact in talimogene laherparepvec, which renders the virussensitive to anti-viral agents such as acyclovir. Therefore, acyclovircan be used to block talimogene laherparepvec replication, if necessary.

Talimogene laherparepvec produces a direct oncolytic effect byreplication of the virus in the tumor, and induction of an anti-tumorimmune response enhanced by the local expression of GM-CSF. In thecontext of melanoma, and other disseminated diseases, this dual activityis beneficial as a therapeutic treatment. The intended clinical effectsinclude the destruction of injected tumors, the destruction of local,locoregional, and distant uninjected tumors, a reduction in thedevelopment of new metastases, a reduction in the rate of overallprogression and of the relapse rate following the treatment of initiallypresent disease, and prolonged overall survival.

Talimogene laherparepvec has been tested for efficacy in a variety of invitro (cell line) and in vivo murine tumor models and has been shown toeradicate tumors or substantially inhibit their growth at dosescomparable to those used in clinical studies. Nonclinical evaluation hasalso confirmed that GM-CSF enhances the immune response generated,enhancing both injected and uninjected tumor responses, and thatincreased surface levels of MIIC class I molecules result from thedeletion of ICP47. Talimogene laherparepvec has been injected intonormal and tumor-bearing mice to assess its safety. In general, thevirus has been well tolerated, and doses up to 1×10⁸ PFU/dose have givenno indication of any safety concerns. (See, for example, Liu et al.,Gene Ther 10: 292-303, 2003).

Clinical studies have been or are being conducted in several advancedtumor types (advanced solid tumors, melanoma, squamous cell cancer ofthe head and neck, breast cancer, and pancreatic cancer), with over 400subjects treated with talimogene laherparepvec (see, for example,Harrington et al., J Clin Oncol. 27(15a):abstract 6018, 2009; Kaufman etal., Ann Surg Oncol. 17: 718-730, 2010; Kaufman and Bines, Future Oncol.6(6): 941-949, 2010). Indeed, talimogene laherparepvec was approved bythe U.S. FDA in October 2015 and by the European Commission in December2015 for the treatment of metastatic melanoma

Inhibition of BRAF Signaling

BRAF gene mutations have been identified in 60% to 70% of malignantmelanomas, 83% of anaplastic thyroid carcinoma, 35% to 69% of papillarythyroid carcinoma, 4% to 16% of colon cancer, 63% of low-grade ovariancarcinoma, 15% of Barrett's esophageal carcinoma, 4% of acute myeloidleukemia, 3-4.8% of head and neck squamous cell carcinoma, 2%-3% ofnon-small-cell lung cancer, 2% of gastric carcinoma, 2% of non-Hodgkin'slymphoma and has been reported in glioma, sarcoma, breast cancer,cholangiocarcinoma, and liver cancer. The combination of oncolytic virus(e.g., talimogene laherparepvec), BRAF inhibitor, and PD-1 inhibitorcombinations of the present invention is particularly useful in treatingtumors comprising BRAF mutations, such as those discussed above.

Thus, in one embodiment, the type of solid malignant tumor to be treatedwith the combination therapy of the present disclosure includes, but isnot limited to, melanoma. In one embodiment, the melanoma comprises aBRAF gene mutation. In other embodiments, additional tumors to betreated with the combination therapy of the present disclosure include,but are not limited to, tumors comprising BRAF gene mutations, includinganaplastic thyroid carcinoma, papillary thyroid carcinoma, colon cancer,ovarian carcinoma, Barrett's esophageal carcinoma, head and necksquamous cell carcinoma, non-small-cell lung cancer, gastric carcinoma,non-Hodgkin's lymphoma, glioma, sarcoma, breast cancer,cholangiocarcinoma, and liver cancer.

Most mutations in BRAF that have been found in human cancers are pointmutations that occur in the kinase domain and are clustered in exons 11and 15 of the gene which contains several regulatory phosphorylationsites (S446, S447, D448, D449, T599, and S602). (Beeram, et al., Journalof Clinical Oncology (2005), 23(27):6771-6790). The most prevalentmutation is the T1799A transversion mutation which accounts for morethan 80% of mutations in the BRAF gene and results in a V600E mutationin B-Raf protein. This mutation is thought to mimic phosphorylation inthe activation segment of B-Raf since it inserts a negatively chargedresidue near two activating phosphorylation sites, T599 and S602, andthus results in constitutively active B-Raf protein in a Ras independentmanner. (Xing, M., Endocrine-Related Cancer (2005), 12:245-262).

Additional mutations of the BRAF gene in human cancer, include but arenot limited to, ARG461ILE, ILE462SER, GLY463GLU, and LYS600GLU(Rajagopalan, H., et al. (Letter) Nature 418: 934, 2002), GLY465VAL andLEU596ARG (Naoki, K., et al., Cancer Res. 62: 7001-7003, 2002), andGLY468ARG, GLY468ALA and ASP593GLY (Lee, J. W., et al., Brit. J Cancer89: 1958-1960, 2003), the references of which are each incorporated byreference in their entirety.

In one embodiment, the oncogenic BRAF to be inhibited in the combinationtherapy of the present disclosure has a mutation selected from the groupconsisting of VAL600GLU (also named as VAL599GLU) (Davies et al. Nature.Jun. 27, 2002; 417(6892):949-54), ARG4611LE, ILE462SER, GLY463GLU, andLYS600GLU (Rajagopalan, H., et al. (Letter) Nature 418: 934, 2002),GLY465VAL and LEU596ARG (Naoki, K., et al., Cancer Res. 62: 7001-7003,2002), and GLY468ARG, GLY468ALA and ASP593GLY (Lee, J. W., et al., Brit.J Cancer 89: 1958-1960, 2003).

Multiple BRAF inhibitors are well known to those of skill in the art.For example, BRAF has been a target of small-molecule therapies to treatcancer (See e.g. Halilovic E, and Solit D B. Curr Opin Pharmacol 2008;8:419-26; and Michaloglou et al., Oncogene 2008; 27:877-95).

In one embodiment of the present disclosure, the inhibitor of oncogenicBRAF is selected from the group consisting of: PLX4720, Sorafenib,RAF265, XL281, AZ628, GSK2118436 (dabrafenib), GDC-0879, and PLX4032(vemurafenib), or a derivative thereof.

In another embodiment, the inhibitor of oncogenic BRAF is selected fromthe group consisting of Sorafenib (Bayer), RAF265 (Novartis), XL281(BMS-908662, Bristol-Myers Squibb; Exelixis), AZ628 (Montagut et al(2008) Cancer Res 68:4853-61), GSK2118436 (dabrafenib), GDC-0879(Selleck Chemicals LLC, Houston, Tex.), PLX4032 (Vemurafenib, Plexxikon,Berkeley, Calif., USA), and PLX4720 (Difluorophenyl-sulfonamine,Plexxikon Inc., Berkeley, Calif., USA).

In yet further embodiments, the inhibitor of oncogenic BRAF is PLX4720(Plexxikon Inc., Berkeley, Calif., USA) (Tsai et al. Proc Natl Acad SciUSA 2008; 105:3041-3046).

The chemical structure of PLX-4720 is shown below:

Immune Checkpoint Inhibitors

Immune checkpoints regulate T cell function in the immune system. Tcells play a central role in cell-mediated immunity. Checkpoint proteinsinteract with specific ligands which send a signal to the T cell andessentially turn off or inhibit T cell function. Cancer cells takeadvantage of this system by driving high levels of expression ofcheckpoint proteins on their surface which results in control of the Tcells expressing checkpoint proteins on the surface of T cells thatenter the tumor microenvironment, thus suppressing the anticancer immuneresponse. As such, inhibition of checkpoint proteins results in completeor partial restoration of T cell function and an immune response to thecancer cells. Examples of checkpoint proteins include, but are notlimited to CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules andis expressed on all NK, γδ, and memory CD8⁺ (αβ) T cells), CD 160 (alsoreferred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR andvarious B-7 family ligands.

Checkpoint inhibitors include any agent that blocks or inhibits in astatistically significant manner, the inhibitory pathways of the immunesystem. Such inhibitors may include small molecule inhibitors or mayinclude antibodies, or antigen binding fragments thereof, that bind toand block or inhibit immune checkpoint receptors or antibodies that bindto and block or inhibit immune checkpoint receptor ligands. Illustrativecheckpoint molecules that may be targeted for blocking or inhibitioninclude, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3,B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2family of molecules and is expressed on all NK, γδ, and memory CD8⁺ (αβ)T cells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2kinases. A2aR and various B-7 family ligands. B7 family ligands include,but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4,B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies, orantigen binding fragments thereof, other binding proteins, biologictherapeutics or small molecules, that bind to and block or inhibit theactivity of one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160 and CGEN-15049. Illustrative immunecheckpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody),anti-OX40, PD-L1 monoclonal Antibody (Anti-B7-H1; MED14736), MK-3475(PD-1 blocker), Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody),BMS-936559 (anti-PDL1 antibody). MPLDL3280A (anti-PDL1 antibody),MSB0010718C (anti-PDL1 antibody) and Yervoy/ipilimumab (anti-CTLA-4checkpoint inhibitor). Checkpoint protein ligands include, but are notlimited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

Programmed cell death protein 1 (PD-1) is a 288 amino acid cell surfaceprotein molecule expressed on T cells and pro-B cells and plays a rolein their fate/differentiation. PD-1 has two ligands, PD-L1 and PD-L2,which are members of the B7 family. PD-1 plays a role in tumor-specificescape from immune surveillance. PD-1 is up-regulated in melanomainfiltrating T lymphocytes (TILs) (Dotti (2009) Blood 114 (8): 1457-58).Tumors have been found to express the PD-1 ligand (PDL-1 and PDL-2)which, when combined with the up-regulation of PD-1 in CTLs, may be acontributory factor in the loss in T cell functionality and theinability of CTLs to mediate an effective anti-tumor response.

Clinical trials in melanoma have shown robust anti-tumor responses withanti-PD-1 blockade. Significant benefit with PD-1 inhibition in cases ofadvanced melanoma, ovarian cancer, non-small-cell lung, prostate,renal-cell, and colorectal cancer have also been described. Studies inmurine models have applied this evidence to glioma therapy. Anti-PD-1blockade adjuvant to radiation promoted cytotoxic T cell population andan associated long-term survival benefit in mice with glioma tumor.

Accordingly, the type of tumor (e.g., solid malignant tumor) to betreated in the combination therapy of the present disclosure includesmelanoma, ovarian cancer, non-small-cell lung carcinoma, prostatecarcinoma, renal-cell carcinoma, and colorectal carcinoma.

One aspect of the present disclosure provides checkpoint inhibitorswhich are antibodies that can act as inhibitors of PD-1, therebymodulating immune responses regulated by PD-1. In one embodiment, theanti-PD-1 antibodies can be antigen-binding fragments. Anti-PD-1antibodies disclosed herein are able to bind to human PD-1 and agonizethe activity of PD-1, thereby inhibiting the function of immune cellsexpressing PD-1. Examples of PD-1 and PD-L1 blockers are described inU.S. Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149,and PCT Published Patent Application Nos: WO03042402, WO2008156712,WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400,and WO2011161699, each of which are incorporated herein by reference.

There are several PD-1 inhibitors currently being tested in clinicaltrials. CT-011 is a humanized IgG1 monoclonal antibody against PD-1. Aphase II clinical trial in subjects with diffuse large B-cell lymphoma(DLBCL) who have undergone autologous stem cell transplantation wasrecently completed. Preliminary results demonstrated that 70% ofsubjects were progression-free at the end of the follow-up period,compared with 47% in the control group, and 82% of subjects were alive,compared with 62% in the control group. This trial determined thatCT-011 not only blocks PD-1 function, but it also augments the activityof natural killer cells, thus intensifying the antitumor immuneresponse.

BMS 936558 is a fully human IgG4 monoclonal antibody targeting PD-1. Ina phase I trial, biweekly administration of BMS-936558 in subjects withadvanced, treatment-refractory malignancies showed durable partial orcomplete regressions. The most significant response rate was observed insubjects with melanoma (28%) and renal cell carcinoma (27%), butsubstantial clinical activity was also observed in subjects withnon-small cell lung cancer (NSCLC), and some responses persisted formore than a year.

BMS 936559 is a fully human IgG4 monoclonal antibody that targets thePD-1 ligand PD-L1. Phase I results showed that biweekly administrationof this drug led to durable responses, especially in subjects withmelanoma. Objective response rates ranged from 6% to 17%) depending onthe cancer type in subjects with advanced-stage NSCLC, melanoma, RCC, orovarian cancer, with some subjects experiencing responses lasting a yearor longer.

MK 3475 is a humanized IgG4 anti-PD-1 monoclonal antibody in Phase IIIstudy alone or in combination with chemotherapy versus chemotherapyalone as first-line therapy for advanced gastric or gastroesophagealjunction (GEJ) adenocarcinoma. MK 3475 is currently undergoing numerousglobal Phase III clinical trials.

MPDL 3280A (atezolizumab) is a monoclonal antibody, which also targetsPD-L1. MPDL 3280A received Breakthrough Therapy Designation from theU.S. Food and Drug Administration (FDA) for the treatment of peoplewhose NSCLC expresses PD-L1 and who progressed during or after standardtreatments.

AMP 224 is a fusion protein of the extracellular domain of the secondPD-1 ligand, PD-L2, and IgG1, which has the potential to block thePD-L2/PD-1 interaction. AMP-224 is currently undergoing phase I testingas monotherapy in subjects with advanced cancer.

Medi 4736 is an anti-PD-L1 antibody that has demonstrated an acceptablesafety profile and durable clinical activity in this dose-escalationstudy. Expansion in multiple cancers and development of MED14736 asmonotherapy and in combination is ongoing.

Thus, in certain embodiments, the PD-1 blockers include anti-PD-1antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS936558, ONO 4538), a fully human IgG4 antibody that binds to and blocksthe activation of PD-1 by its ligands PD-L1 and PD-L2;pembrolizumab/lambrolizumab (MK-3475 or SCH 900475), a humanizedmonoclonal IgG4 antibody against PD-1: CT-011 a humanized antibody thatbinds PD-1; AMP-224 is a fusion protein of B7-DC: an antibody Fcportion; BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blockade. Otherimmune-checkpoint inhibitors include lymphocyte activation gene-3(LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein(Brignone et al., 2007, J. Immunol. 179:4202-4211). Otherimmune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 andB7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo etal., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM3(T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcadeet al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J.Exp. Med. 207:2187-94).

Pharmaceutical Compositions and Administration

The oncolytic virus (e.g., talimogene laherparepvec), a BRAF inhibitor,and an immune checkpoint inhibitor, as disclosed herein can beadministered to a patient by various routes including, for example,orally or parenterally, such as intravenously, intramuscularly,subcutaneously, intraorbitally, intracapsularly, intraperitoneally,intrarectally, intracisternally, intratumorally, intravasally,intradermally or by passive or facilitated absorption through the skinusing, for example, a skin patch or transdermal iontophoresis,respectively. The oncolytic virus (e.g., talimogene laherparepvec), aBRAF inhibitor, and an immune checkpoint inhibitor can also beadministered to the site of a pathologic condition, for example,intravenously or intra-arterially into a blood vessel supplying a tumor.In one embodiment, the oncolytic virus (e.g., talimogene laherparepvec)is injected into the tumor (i.e., via intratumoral injection). In oneembodiment, the BRAF inhibitor is administered orally. And, in oneembodiment, the anti-PD-1 compound (e.g., antibody) is administeredsystemically (e.g., intravenously).

Thus, as discussed herein, the pharmaceutical formulations of thepresent invention may contain one, two, or all three of: an oncolyticvirus (e.g., talimogene laherparepvec), a BRAF inhibitor, and ananti-PD-1 compound. Such pharmaceutical compositions can be used in themethods described here as appropriate. For example, in some embodimentsof the methods described herein, the oncolytic virus (e.g., talimogenelaherparepvec) is injected into the tumor, the BRAF inhibitor isadministered orally, and the anti-PD-1 compound (e.g., antibody) isadministered intravenously. As would be appreciated by those skilled inthe field, the order of administration can be varied as appropriate.

The total amount of The oncolytic virus (e.g., talimogenelaherparepvec), BRAF inhibitor, and immune checkpoint inhibitor can beadministered to a subject as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which multiple doses areadministered over a prolonged period of time.

Determining the dosage and duration of treatment according to any aspectof the present disclosure is well within the skills of a professional inthe art. Those skilled in the field are readily able to monitor patientsto determine whether treatment should be started, continued,discontinued or resumed at any given time. For example, dosages of thecompounds are suitably determined depending on the individual casestaking symptoms, age and sex of the subject and the like intoconsideration. The amount of the compound to be incorporated into thepharmaceutical composition of the present disclosure varies with dosageroute, solubility of the compound, administration route, administrationscheme and the like. An effective amount for a particular patient mayvary depending on factors such as the condition being treated, theoverall health of the patient and the method, route and dose ofadministration. The clinician using parameters known in the art makesdetermination of the appropriate dose. Generally, the dose begins withan amount somewhat less than the optimum dose and it is increased bysmall increments thereafter until the desired or optimum effect isachieved. Suitable dosages can be determined by further taking intoaccount relevant disclosure in the known art.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. The antibody molecules can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration isintravenous injection or infusion. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. In certain embodiments, the active compoundmay be prepared with a carrier that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical Compositions and Administration of TalimogeneLaherparepvec

Currently, talimogene laherparepvec is administered via intralesionalinjection. Clinical studies have demonstrated that talimogenelaherparepvec can be injected directly into cutaneous, subcutaneous ornodal lesions that are visible, palpable, or can be injected withultrasound-guidance. Talimogene laherparepvec is provided in 1 mLsingle-use vials in fixed dosing concentrations: 10⁶ pfu/mL for initialdosing and 10⁸ pfu/mL for subsequent dosing (Reske, et al. J Immunol,2008, 180(11): p. 7525-36). The volume that is injected may varydepending on the tumor type. For example, talimogene laherparepvec isadministered by intratumoral injection into injectable cutaneous,subcutaneous, and nodal tumors at a dose of up to 4.0 ml of 10⁶ plaqueforming unit/mL (PFU/mL) at day 1 of week 1 followed by a dose of up to4.0 ml of 10⁸ PFU/mL at day 1 of week 4, and every 2 weeks (±3 days)thereafter. The recommended volume of talimogene laherparepvec to beinjected into the tumor(s) is dependent on the size of the tumor(s) andshould be determined according to the injection volume guideline inTable 1 (and as shown in patent application PCT/US2013/057542).

TABLE 1 Talimogene Laherparepvec Injection Volume Guidelines Based onTumor Size Tumor Size (longest dimension) Maximum Injection Volume ≥5.0cm 4.0 ml >2.5 cm to 5.0 cm 2.0 ml >1.5 cm to 2.5 cm 1.0 ml >0.5 cm to1.5 cm 0.5 ml ≤0.5 cm 0.1 mlAll reasonably injectable lesions should be injected with the maximumdosing volume available on an individual dosing occasion. On eachtreatment day, prioritization of injections is recommended as follows:any new injectable tumor that has appeared since the last injection; bytumor size, beginning with the largest tumor; any previouslyuninjectable tumor(s) that is now injectable.

Pharmaceutical Compositions and Administration of BRAF Inhibitor

In certain embodiments, a BRAF inhibitor is administered in an amount ofabout 0.1 mg/day to about 1200 mg/day, about 1 mg/day to about 100mg/day, about 10 mg/day to about 1200 mg/day, about 10 mg/day to about100 mg/day, about 100 mg/day to about 1200 mg/day, about 400 mg/day toabout 1200 mg/day, about 600 mg/day to about 1200 mg/day, about 400mg/day to about 800 mg/day or about 600 mg/day to about 800 mg/day.

In other embodiments, total daily dose of a BRAF inhibitor is selectedfrom about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 30 mg,about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg,about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg,about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg,about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg,about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg,about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg,about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg,about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg,about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg,about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg,about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg,about 580 mg, about 590 mg, about 600 mg, about 650 mg, about 700 mg,about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg,about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg,about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100 mg, about2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg,about 2400 mg, about 2450 mg, about 2500 mg, about 2550 mg, about 2600mg, about 2650 mg, about 2700 mg, about 2750 mg, about 2800 mg, about2850 mg, about 2900 mg, about 2950 mg, or about 3000 mg.

Pharmaceutical Compositions and Administration of Immune CheckpointInhibitor

The immune checkpoint inhibitor is administered in the form of acomposition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Thecompositions may comprise one or more substances selected from the groupconsisting of a buffer, an antioxidant such as ascorbic acid, a lowmolecular weight polypeptide (such as those having fewer than 10 aminoacids), a protein, an amino acid, a carbohydrate such as glucose,sucrose or dextrins, a chelating agent such as EDTA, glutathione, astabilizer, and an excipient. Neutral buffered saline or saline mixedwith specific serum albumin are examples of appropriate diluents. Inaccordance with appropriate industry standards, preservatives such asbenzyl alcohol may also be added. The composition may be formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents. Suitable components are nontoxic to recipients at the dosagesand concentrations employed.

In certain embodiments, the checkpoint inhibitor is administered in 0.01mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.7mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, or any combination thereof doses. In certainembodiments the checkpoint inhibitor is administered once a week, twicea week, three times a week, once every two weeks, or once every month.In certain embodiments, the checkpoint inhibitor is administered as asingle dose, in two doses, in three doses, in four doses, in five doses,or in 6 or more doses.

In certain embodiments, the anti-PD-1 antibody is administered byinjection (e.g., subcutaneously or intravenously) at a dose of about 1to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., oncea week to once every 2, 3, or 4 weeks. In one embodiment, theanti-PD-antibody is administered at a dose from about 10 to 20 mg/kgevery other week.

In one embodiment, the anti-PD-1 antibody molecule, e.g., nivolumab, isadministered intravenously at a dose from about 1 mg/kg to 3 mg/kg,e.g., about 1 mg/kg, 2 mg/kg or 3 mg/kg, every two weeks. In oneembodiment, the anti-PD-1 antibody molecule, e.g., nivolumab, isadministered intravenously at a dose of about 2 mg/kg at 3-weekintervals. In one embodiment, nivolumab is administered in an amountfrom about 1 mg/kg to 5 mg/kg, e.g., 3 mg/kg, and may be administeredover a period of 60 minutes, ca. once a week to once every 2, 3 or 4weeks.

EXAMPLES Materials and Methods

BRAF^(F-V600E/+); PTEN^(F−/−); Tyr::CreERT2 Mouse Model

All animal experiments were performed in accordance with institutionaland national guidelines and were approved by the Institutional AnimalCare and Use Committee at Columbia University. Mice were purchased fromJackson Laboratories (https://www.jax.org/strain/013590) and bred todesired genotype. The inducible mouse melanoma model was obtained bycrossing the C57BL/6J Tyr::CreERT2 (homo- or heterozygotic), C57BL/6JPTENF−/− (homozygotic), and mixed-background BRAF F-V600E/_(heterozygotic) mouse strains. Mice were genotyped at 7 days of age.Mice heterozygous BRAF (BRAF^(F-V600E/+)), homozygous for fleoxed PTEN(PTEN^(F−/−)) and Tyr::CreERT2 were kept for study. Hlomozygous and wildtype mice were kept for breeding.

Tumor Induction

Tumors were induced on the skin of the melanoma model mice as describedpreviously (Dankort et al. Nat Genet. 2009 May; 41(5):544-52). In brief,mice were anesthetized using isoflurane (Henry Schein Animal Health,Dublin, Ohio) and then 2 aliquots of 5 μL of 10 mg/mL 4-hydroxytamoxifen(4-TH) ≥70% Z isomer (remainder primarily E-isomer) (Sigma-Aldrich, St.Louis, Mo.) dissolved in pure ETOH (Fisher Scientific, Fair Lawn. N.J.)was topically applied to the shaven right flank of 6 to 8-week-old micefor three consecutive days. Tumor outgrowth was monitored twice weekly,from 4 weeks following tumor induction.

Animal Treatment

Tumors developed 4-5 weeks post induction and mice with tumors between5-60 mm³ of volume (calculated by (L×W×H×π)/6) (or >5 mm in diameter)were randomized into 6 possible treatment groups to treat 10 mice/group(FIGS. 5 and 6). Mice assigned to OncoVex^(mGM-CSF) treatment groupswere transferred to an Animal Biosafety Level (ASBL) 2 facility.Treatments were administered through mouse chow, intraperitonealinjection, and intratumoral injection. Control chow (Research Diets. NewBrunswick, N.J.) or BRAFi chow (Research Diets, New Brunswick, N.J.)containing the drug PLX 4720 (Selleck Chemicals, Houston, Tex.), wasgiven at start of study and continuously replenished. Intraperitonealinjections of 100 μL of Isotype Control 2A3 (Bio X Cell, West Lebanon,N.H.) or anti-PD-1 of (Bio X Cell, West Lebanon, N.H.) diluted to 1:1ratio with PBS (MP Biomedicals, LLC, Solon, Ohio) were given three daysa week, every other week. Intratumoral injections of 100 μL of PBS or 50uL of 1×10⁷ pfu of OncoVex^(mGM-CSF) (Amgen, Thousand Oaks, Calif.)diluted with 50 μL additional PBS were given biweekly. Each tumorreaching volume requirement were injected. Tumors were measuredbiweekly. Weight was measured once a week. Treatment was stopped in micewhen there was suspected toxicity, the tumor appeared necrotic, themouse appeared debilitated, or weight loss >5%. End of study wasdetermined by tumor volume ≥3000 mm³, ≥25% of the tumor ulcerated, orthe mouse had ≥20% weight loss or other signs of debilitation.

A general scheme describing the use of these mice in these Examples isoutlined in FIG. 1.

Immune Monitoring

For analysis of tumor-infiltrating leukocytes, mice bearing tumors ≥3000mm3 (newly scored dead on survival curve) were sacrificed by CO₂inhalation, confirmed with neck dislocation, and tumors, spleen andlymph nodes were dissected. The tumor mass was homogenized, digestedwith 0.4 mg/mL collagenase (Sigma-Aldrich, St. Louis, Mass.), incubatedat 37° C. for 20 minutes, further homogenized with a Gentle MACSDissociator (Miltenyi Biotec, Inc., San Francisco, Calif.) and thenstrained through 40-m nylon filters (Fisher Scientific, Pittsburgh,Pa.). Spleen was cut and cells collected per protocol using RBC Celllysis and straining. Lymph node cells were collected per protocol bycutting and straining. Cells were incubated with antibodies for 15minutes on ice, fixed with fixation/permeabilitzation for 30 minutes,then incubated with the intracellular antibody FoxP3 for 30 minutes.Cells were then acquired with the BDFortessa Flow Cytometer within twoweeks of fixation and analyzed with FlowJo V10 (FlowJo, LLC, Ashland,Oreg.). Fluorochrome conjugated antibodies specific for mouse CD45(clone 30-F11) (eBioscience Inc., San Diego, Calif.), CD3 (17A2)(Biolegend, San Diego, Calif.), CD4 (clone RM4-5) (BD Biosciences, SanJose, Calif.), CD8 (clone 53-6.7) (Biolegend, San Diego, Calif.), PD-1(clone J43) (BD Biosciences. San Jose, Calif.), NK1.1 (clone PK136)(eBioscience Inc., San Diego, Calif.), FOXP3 (clone NRRF-20)(eBiosciences Inc., San Diego, Calif.), and fixable viability dye(eFlour 506) (eBiosciences Inc., San Diego, Calif.) were used forstaining.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6 (GraphPadSoftware, Inc., La Jolla, Calif.). Survival analysis was performed usingthe log-rank (Mantel-Cox) test with Bonferroni correction. Significancewas defined at a P value of <0.05, using a nonparametric Mann-Whitney ttest. Bonferroni correction was used for multigroup comparisons. P-valuewas calculated as compared to control group. Multiplex IHC cell densityand nearest neighbor analysis will be performed using Inform Software(Perkin Elmer, Waltham, Mass.).

General Methods

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology. Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science.Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,NY, pp. 16.0.5-16.22.17: Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protocols in Immunology.Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor. N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Example 1 Generation of the BRAF^(F-V600); PTEN^(F−/−); Tyr::CreERT2Mouse Model

One of the most common genetic alterations in human melanoma is a pointmutation in the BRAF proto-oncogene. In addition to BRAF mutation, theprogression of malignant melanoma is also driven by loss of expressionof tumor suppression genes, such as phosphatase and tensin homolog(PTEN). In order to build a model that reflects both initiation andprogression phases of melanoma, Dankort et al. have generated aconditional mouse model of BRAF^(V600) induced, PTEN deficientmetastatic human melanoma, which allows for the controlled timing andlocation of melanoma initiation (Nat Genet. 2009 May; 41(5):544-52). Asmost studies related to immunotherapy research have been conducted usingC57BL/6J mice, the BRAF^(F-V600); PTEN^(F−/−); Tvr::CreERT2 induciblemelanoma model was crossed on a C57BL/6J genetic background. Previousstudies have demonstrated that C57BL/6J Tyr::CreER;PTEN(^(F−/−));BRAF(^(F-V600E/+)) melanoma model can be used as a standard model inwhich targeted therapy combinations can be tested in a high-throughputmanner (Hlooijkaas et al. Am J Pathol. 2012 September; 181(3):785-94).

Following generation of BRAF^(F-V600); PTEN^(F−/−); Tyr::CreERT2animals, tumor formation was induced as described previously (Dankort etal. Nat Genet. 2009 May; 41(5):544-52).

Example 2 Combined Therapy Using OncoVex^(mGM-CSF), BRAF Inhibition, andImmune Checkpoint Blockade Leads to Prevention/Establishment of TumorInduction

Melanomas are resistant to both radiotherapy and chemotherapy. Previousstudies have demonstrated tumor reduction when melanomas (developedusing a spontaneous model described herein) were treated withcombination therapy using OncoVex^(mGM-CSF) and a BRAF inhibitor. Inorder to improve response to therapy, the effects of immune checkpointblockade on tumor formation when combined with administration ofOncoVex^(mGM-CSF) and BRAF inhibitor were tested.

BRAF^(F-V600); PTEN^(F−/−); Tyr::CreERT2 animals were divided into 6groups, where tumor formation was induced using 4-HT. 4-5 weeksfollowing tumor induction, each of the 6 groups received a differenttreatment: (1) group 1 received control chow, intraperitoneal injectionof isotype control 2A3, and intratumoral injection of PBS; (2) group 2received BRAF inhibitor PLX 4720 containing chow (BRAFi chow),intraperitoncal injection of isotype control 2A3, and intratumoralinjection of PBS; (3) group 3 received BRAFi chow, intraperitonealinjection of anti-PD-1 antibody, and intratumoral injection of PBS; (4)group 4 received BRAFi chow, intraperitoneal injection of isotypecontrol 2A3, and intratumoral injection of OncoVex^(mGM-CSF); (5) group5 received BRAFi chow, intraperitoneal injection of anti-PD-1 antibody,and intratumoral injection of OncoVex^(mGM-CSF); and (6) group 6received control chow, intraperitoneal injection of anti-PD-1 antibody,and intratumoral injection of OncoVex^(mGM-CSF). Tumor volume wasmeasured bi-weekly.

As shown in FIG. 2, addition of immune checkpoint inhibitor (such asanti-PD-1 antibody) to OncoVex^(mGM-CSF) (F and BRAF inhibitioncombination therapy prevents/inhibits tumor formation significantly whencompared to all other treatment groups. Animals treated with tamoxifen(4HT) for 35 days while receiving triple combination therapy (BRAFichow, anti-PD-1 antibody, and OncoVex^(mGM-CSF); group 5) did notdevelop tumors, while the control group established tumors (group 1)exceeding 1700 mm³ in size. Incorporating anti-PD-1 antibody into thetreatment regimen consisting of OncoVex^(mGM-CSF) and BRAF inhibitionsignificantly improved a therapeutic effect of combinedOncoVex^(mGM-CSF) and BRAF inhibition therapy, where OncoVex^(mGM-CSF)and BRAF inhibition combination therapy resulted in tumors. Theseresults indicate that inclusion of an immune checkpoint inhibitor intoOncoVex^(mGM-CSF) and BRAF inhibition combination therapy substantiallyimproves in vivo treatment outcome.

These results were further confirmed in FIGS. 4A and 4B. Here, tumorvolume was calculated for each mouse and the mean of the tumor volumesof the mice in each group was plotted on a tumor growth curve up to 33days (FIG. 4A) and mean tumor volumes of the mice in groups receivingBRAFi chow is plotted on a tumor growth curve up to 57 days (FIG. 4B).Group 5 mice (BRAFi chow, intraperitoneal injection of anti-PD-1antibody, and intratumoral injection of OncoVex^(mGM-CSF)) hadsignificantly smaller tumor volume than group 2 mice (BRAFi alone)(p=0.0141) (FIG. 4B). Mice were sacrificed at end of study which wasdetermined by tumor volume of 3000 mm³ or greater, or greater than 2.6cm in a single dimension, more than 25% of the tumor was ulcerated, themouse had >20% weight loss, or other signs of debilitation.

Example 3 Combined Therapy Using OncoVex^(mGM-CSF), BRAF Inhibition, andImmune Checkpoint Blockade Leads to Tumor Regression

The inclusion of an immune checkpoint inhibitor was tested incombination with OncoVex^(mGM-CSF) and BRAF inhibition therapy todetermine whether this triple therapy can stimulate regression or slowdown a progression of preexisting tumors in mice. To do so, theBRAF^(F-V600); PTEN^(F−/−); Tyr::CreERT2 animals were divided into 6groups, where tumor formation was induced using 4-HT. Four-five weeksfollowing tumor induction, when the animals had measurable melanomatumors, each of the 6 groups received a different treatment: (1) group 1received control chow, intraperitoneal injection of isotype control 2A3,and intratumoral injection of PBS; (2) group 2 received BRAF inhibitorPLX 4720 containing chow (BRAFi chow), intraperitoneal injection ofisotype control 2A3, and intratumoral injection of PBS; (3) group 3received BRAFi chow, intraperitoneal injection of anti-PD-1 antibody,and intratumoral injection of PBS; (4) group 4 received BRAFi chow,intraperitoneal injection of isotype control 2A3, and intratumoralinjection of OncoVex^(mGM-CSF); (5) group 5 received BRAFi chow,intraperitoneal injection of anti-PD-1 antibody, and intratumoralinjection of OncoVex^(mGM-CSF); and (6) group 6 received control chow,intraperitoneal injection of anti-PD-1 antibody, and intratumoralinjection of OncoVex^(mGM-CSF). Tumor volume was measured bi-weekly.

Animals were monitored and mouse survival was plotted using Kaplan-Meiersurvival curve (FIG. 3). As shown in FIG. 3, animals in the controlgroup succumbed to disease as early as 40 days following the start oftreatment (Line 1), while animals receiving both OncoVex^(mGM-CSF) andBRAF inhibitor PLX 4720 remained alive for up to 130 days (Line 4).There was a statistically significant difference when PD-1 inhibitionwas included into the treatment regimen. As shown in FIG. 3, significantimprovement in survival, was observed when animals received BRAFi chow,intraperitoneal injection of anti-PD-1 antibody, and intratumoralinjection of OncoVex^(mGM-CSF), as manifested by the fact that a numberof animals survived past 130 days (Line 5).

These results were further confirmed in FIGS. 4C and 4D. Survival ofmice in each treatment group was plotted on a KM curve. Survival of micein group 2 (BRAFi alone) (p<0.0001), group 3 (BRAFi+α-PD1) (p=0.0002),group 4 (BRAFi+OncoVex^(mGM-CSF)) (p<0.0001), and group 5(BRAFi+α-PD1+OncoVex^(mGM-CSF)) (p<0.0001) was significantly longer whencompared to group 1 (control) (FIG. 4C). Survival of mice in groupsreceiving BRAFi was also plotted using a KM curve and shows a trendtowards significance in group 4 (p=0.1218) and group 5 (p=0.1561) whencompared to group 2 (BRAFi alone) (FIG. 4D). When more than 10% of thetumor was ulcerated, intratumoral injections were stopped. Whencomparing the number of mice whose treatment was stopped due toulceration, findings showed a higher number of these mice were in groupsreceiving OncoVex^(mGM-CSF) while BRAFi and anti-PD1 did not affectulceration to the same extent (FIG. 4E). The size of the tumors of themice in each group when intratumoral injections were stopped was alsoplotted and found that the tumor size of mice in group 5 (p=0.0214) andgroup 6 (p=0.0074) were significantly smaller than the tumor size ofgroup 1 (control) mice (FIG. 4F).

These experiments demonstrate that incorporation of an immune checkpointinhibitor into OncoVex^(mGM-CSF) and BRAF inhibition combination therapysubstantially improves survival of melanoma inflicted animals, whichserve as a well-accepted model for human disease.

Example 4 Combined Therapy Using OncoVex^(mGM-CSF), BRAF Inhibition, andImmune Checkpoint Blockade Results in Decreased CD4+Foxp3+ Tregs TumorInfiltration

The effect of combined OncoVex^(mGM-CSF), BRAF inhibition, and immunecheckpoint blockade therapy on intratumoral CD4+Foxp3+ Tregsinfiltration is also analyzed herein. As shown in FIG. 5, the percentageof CD4+Foxp3+ Tregs was significantly lower in cells receiving anti-PD-1therapy in addition to OncoVex^(mGM-CSF) and BRAF inhibition, comparedwith those that received only OncoVex^(mGM-CSF) and BRAF inhibitioncombined therapy.

These results were further confirmed in FIG. 6. Flow cytometry wasperformed on tumor, spleen and lymph nodes of each mouse for analysis oftumor-infiltrating lymphocytes (TILs). Cells were stained with CD45,CD3, CD4, CD8, PD1, NK1.1, FOXP3, and fixable viability dye. The percentofCD8+/CD3+(Cytotoxic T lymphocytes (CTLs)) was significantly higher ingroup 3 (BRAFi+α-PD1) (p=0.0476), group 4 (BRAFi+OncoVex^(mGM-CSF))(p=0.0004), group 5 (BRAFi+α-PD1+OncoVex^(MCSF)) (p=0.0001), and group 6(α-PD1+OncoVex^(mGM-CSF)) (p=0.0001) when compared to group 1 (control)(FIG. 6A). CTL infiltration was highest in groups receivingOncoVex^(mGM-CSF) when compared to groups not receivingOncoVex^(mGM-CSF) (p<0.0001) (FIG. 6B). The percent ofCD4+FOXP3+/CD4+(T-regulatory cells (Tregs)) is decreased in groups 4(p=0.0082), 5 (p<0.0001), and 6 (p=0.0031) when compared to group 1(control). (FIG. 6C). Treg infiltration is lowest in groups receivingOncoVex^(mGM-CSF) CSF when compared to groups not receivingOncoVex^(mGM-CSF) (p<0.0001) (FIG. 6D). No trends were found in thespleen and lymph nodes.

Example 5 Analysis of Tumor Samples Exposed to Combined TherapyIncluding OncoVex^(mGM-CSF), BRAF Inhibition, and Immune CheckpointBlockade

Tumor tissue was zinc fixed and paraffin embedded at end of study toperform quantitative multiplex immunofluorescence (qmIF) using Vectra™(Perkin Elmer), a quantitative pathology workstation, allowing forevaluation of density and spatial relationships of immune cells. Tumortissue from 36 mice was stained for qmIF (6 mice from each treatmentgroup) for CD3, CD4, CD8, FOXP3, PDL1, F4/80, and DAPI. Multispectralimages of these slides are obtained and are analyzed using inForm™software (Perkin Elmer).

6 mice are enrolled in each of the same 6 treatments groups (total of 36mice) and tumors are analyzed from sacrificed mice at 30 days oftreatment. Tumor tissues from these mice are formalin-fixedparaffin-embedded (FFPE) for NanoString analysis, to profileimmune-related genes, as well as confirm infection of OncoVex^(mGM-CSF),using the Mouse Immunology Panel and spiking in early, immediate early,and late HSV genes. 13 mice have completed this portion of the study. 12mice are currently enrolled and the additional 11 mice are being bredand induced.

In other experiments, multiplex immunohistochemistry analyses areperformed on slides cut from paraffin embedded blocks with dissectedtumors from treatment mice. The slides are stained with antibodies forCD3, CD8, HSV, BRAF, FoxP3, and CD4. This panel is optimized and finalantibodies are determined once of optimization is complete.

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All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g. Genbanksequences or GenelD entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicants,pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g. Genbank sequences or GeneIDentries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. 1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.The entire disclosure of each of the patent documents, includingcertificates of correction, patent application documents, scientificarticles, governmental reports, websites, and other references referredto herein is incorporated by reference herein in its entirety for allpurposes. In case of a conflict in terminology, the presentspecification controls.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are to be considered in all respects illustrative ratherthan limiting on the invention described herein. Further, it should beunderstood that the order of steps or order for performing certainactions is immaterial so long as the invention remains operable.Moreover, two or more steps or actions can be conducted simultaneously.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the case of conflict, thepresent specification will control.

What is claimed is:
 1. A method for treating a solid malignant tumor ina subject comprising: administering to the subject a therapeuticallyeffective amount of BRAF inhibitor, an oncolytic virus, and an immunecheckpoint inhibitor.
 2. The method of claim 1, wherein the oncolyticvirus is an HSV-1-based oncolytic virus.
 3. The method of claim 1,wherein the oncolytic virus is talimogene laherparepvec.
 4. The methodof claim 1, wherein the immune checkpoint inhibitor is selected from asmall molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, and combinationsthereof.
 5. The method of claim 1, wherein the immune checkpointinhibitor is a PD-1 antagonist.
 6. The method of claim 5, wherein theimmune checkpoint inhibitor is an anti-PD-1 antibody.
 7. The method ofclaim 1, wherein the administration of the immune checkpoint inhibitoris continued until it induces tumor regression, remission, oreradication.
 8. The method of claim 1, wherein the administration ofeach BRAF inhibitor, oncolytic virus, and the immune checkpointinhibitor is continued until it induces tumor regression, remission, oreradication.
 9. The method of claim 1, wherein the BRAF inhibitor isselected from the group consisting of PLX4720, Sorafenib, RAF265, XL281,AZ628, GSK2118436 (dabrafenib), GDC-0879, and PLX4032 (vemurafenib), ora derivative thereof.
 10. The method of claim 1, wherein the solidmalignant tumor is melanoma.
 11. A method for treating melanoma in asubject comprising: administering to the subject a therapeuticallyeffective amount of a BRAF inhibitor, an oncolytic virus, and an immunecheckpoint inhibitor.
 12. The method of claim 11, wherein the oncolyticvirus is an HSV-1-based oncolytic virus.
 13. The method of claim 11,wherein the oncolytic virus is talimogene laherparepvec.
 14. The methodof claim 11, wherein the immune checkpoint inhibitor is selected from asmall molecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, and combinationsthereof.
 15. The method of claim 11, wherein the immune checkpointinhibitor is a PD-1 antagonist.
 16. The method of claim 11, wherein theimmune checkpoint inhibitor is an anti-PD-1 antibody.
 17. The method ofclaim 11, wherein the administration of the checkpoint inhibitor iscontinued until it induces melanoma tumor regression, remission, oreradication.
 18. The method of claim 11, wherein the administration ofeach BRAF inhibitor, oncolytic virus, and checkpoint inhibitor iscontinued until it induces melanoma tumor regression, remission, oreradication.
 19. The method of claim 11, wherein the BRAF inhibitor isselected from the group consisting of PLX4720, Sorafenib, RAF265, XL281,AZ628, GSK2118436 (dabrafenib), GDC-0879, and PLX4032 (vemurafenib), ora derivative thereof.
 20. A method of reducing CD4+FOXP3+ cellularpopulation within a solid malignant tumor, the method comprisingdelivering to the tumor an effective amount of a BRAF inhibitor, anoncolytic virus, and a checkpoint inhibitor.
 21. The method of claim 20,wherein the immune checkpoint inhibitor is selected from a smallmolecule, protein, peptide, antibody or antigen binding fragmentthereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049 and combinationsthereof.
 22. The method of claim 20, wherein the immune checkpointinhibitor is a PD-1 antagonist.
 23. The method of claim 20, wherein theimmune checkpoint inhibitor is an anti-PD-1 antibody.
 24. The method ofclaim 20, wherein BRAF inhibitor is selected from the group consistingof PLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436 (dabrafenib),GDC-0879, and PLX4032 (vemurafenib), or a derivative thereof.
 25. Amethod of stimulating an immune response in a subject comprising:administering to the subject a therapeutically effective amount of aBRAF inhibitor, an oncolytic virus, and a checkpoint inhibitor.
 26. Themethod of claim 25, wherein the immune checkpoint inhibitor is selectedfrom a small molecule, protein, peptide, antibody or antigen bindingfragment thereof, directed against CTLA-4, PD-L1, PD-L2, PD-1, BTLA,HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, andcombinations thereof.
 27. The method of claim 25, wherein the immunecheckpoint inhibitor is a PD-1 antagonist.
 28. The method of claim 25,wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. 29.The method of claim 25, wherein the administration of each BRAFinhibitor, oncolytic virus, and checkpoint inhibitor is continued untilstimulation of the immune response is achieved.
 30. The method of claim25, wherein the BRAF inhibitor is selected from the group consisting ofPLX4720, Sorafenib, RAF265, XL281, AZ628, GSK2118436 (dabrafenib),GDC-0879, and PLX4032 (vemurafenib), or a derivative thereof.